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Título: | Reactivation of lipases by the unfolding and refolding of covalently immobilized biocatalysts |
Autor: | Rueda, Nazzoly; Sousa dos Santos, José Cleiton; Torres Sáez, Rodrigo; Barbosa, Oveimar CSIC ORCID; Ortiz, Claudia; Fernández-Lafuente, Roberto CSIC ORCID | Fecha de publicación: | 2015 | Editor: | Royal Society of Chemistry (UK) | Citación: | RSC Advances 5(68): 55588-55594 (2015) | Resumen: | Lipases from Candida antarctica (isoform B) (CALB) and Thermomyces lanuginosus (TLL) have been immobilized either covalently or by interfacial activation versus an octyl support, followed by covalent attachment by glyoxyl groups using octyl–glyoxyl agarose beads (OCGLX). These biocatalysts have been submitted to successive cycles of unfolding by incubation in 9 M guanidine and refolding by incubation in aqueous 100 mM phosphate buffer at pH 7, before and after total inactivation in the presence of organic solvents. The four preparations have been reactivated to some extent using this strategy, but the results depended on the method of preparation. Glyoxyl-immobilized CALB may recover 100% of its activity versus p-nitrophenyl butyrate, but after solvent inactivation the recovery of activity was reduced to 95%. The pure covalent TLL preparation recovered around 80% of its activity, either before or after solvent inactivation. Both enzymes showed less recovery of activity using OCGLX, as might be expected from the hydrophobic nature of the supporting groups (60% for CALB and 45% for TLL). In addition, using enzymes previously inactivated by solvent, recovery decreased by 5–10%. These values were maintained through three successive cycles. However, using R- and S-methyl mandelate, it was clear that recovery of activity decreased with further reactivation cycles. Taken as a whole, the unfolding and refolding effect may be used to recover a degree of enzyme activity. This is relevant in terms of application, as it may allow the enzyme preparations to be used for a longer period. However, to reach a similar enzyme structure in each reactivation cycle, it will be necessary to undertake further studies involving the use of other supports in order to improve the unfolding and refolding steps. | Versión del editor: | https://doi.org/10.1039/C5RA07379K | URI: | http://hdl.handle.net/10261/190777 | DOI: | 10.1039/c5ra07379k | E-ISSN: | 2046-2069 |
Aparece en las colecciones: | (ICP) Artículos |
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